Robotic inside-out patch clamp technique for monitoring vesicle rupture

Abstract

The present invention discloses a robotic inside-out patch clamp technique based on the detection of vesicle rupture process, which relates to the field of cell-level micromanipulation technology, and specifically includes S1: by analyzing the mechanism of vesicle formation and rupture, a method for sealing a monolayer membrane patch is obtained by using a reverse verification method, thereby verifying that a monolayer membrane patch with the inside facing out is obtained; S2: a fluid exchange device is designed to ensure the rapidity and stability of the fluid exchange process; S3: a monolayer membrane patch with the inside facing out is obtained by robotic operation. The present invention completes the robotic inside-out monolayer membrane patch on adherent cells for the first time through the detection of the vesicle rupture process and the design of a rapid fluid exchange device, which is of great significance for promoting the promotion and application of the inside-out patch clamp technique.

Description

Technical Field

[0001] The present invention relates to the field of cell-level micromanipulation technology, and in particular to a robotic inside-out patch clamp technology based on vesicle rupture process detection. Background Art

[0002] The patch clamp technique was invented by German scientists Neher and Sakmann in 1976, and they won the 1991 Nobel Prize in Medicine and Physiology for this. The patch clamp uses a glass microtube electrode of micrometer size to contact the cell surface, suck part of the cell membrane into the microtube, and form a GΩ-level high-resistance seal (hereinafter referred to as high-resistance seal) between the cell membrane and the inner wall of the microtube to suppress electromagnetic noise. Further, the sucked cell membrane patch is peeled off from the cell for detection. Since the area of ​​the peeled cell membrane patch is only square micrometers, and the number of ion channels contained on it is usually in the single digit, the patch clamp has the ability to detect the electrical signals of single-channel ions of a single cell, and is known as the "gold standard" for cell ion channel research. The most basic work in brain science and neuroscience research is to explore the function of neurons by detecting the electrical signals of single-channel ions of nerve cells. Therefore, the current patch clamp technique has been widely used in brain science and neuroscience research.

[0003] According to the different orientations of the membrane patch after peeling, the operation mode of single-channel patch clamp is divided into two types: inside-out and outside-out. Among them, the inside-out patch clamp technique is to quickly pull the absorbed cell membrane to separate it from the cell to form a vesicle after the high-resistance seal is formed, and then the vesicle is ruptured by immersion in a low-calcium solution or exposure to air to form an inside-out cell membrane patch for measurement. The inside-out method is an essential technology for detecting single ion channel electrophysiological signals. However, the manual inside-out membrane patch production requires extremely high professional level of the operator, and only a very small number of top operators in individual laboratories around the world can complete it. Improving the efficiency of the inside-out mode through robotic operation and lowering the technical threshold of the inside-out patch clamp are of great significance to improving the overall success rate and measurement accuracy of the patch clamp technology and promoting the promotion and application of the patch clamp technology in brain science and neuroscience. Summary of the invention

[0004] The object of the present invention is to provide a robotized inside-out patch clamp technology based on vesicle rupture process detection to solve the problems of high difficulty and high professional level requirements in the existing manual inside-out operation mode mentioned in the background technology.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme:

[0006] The present invention provides a robotic inside-out patch clamp technique based on vesicle rupture process detection, comprising the following steps:

[0007] S1: By analyzing the mechanism of vesicle formation and rupture, the reverse verification method was used to obtain a method for sealing the monolayer membrane, thereby verifying that a monolayer membrane with the inside facing out was obtained.

[0008] The process of forming the inside-out operation mode is as follows Figure 1 As shown, it is carried out on the basis of the cell attachment mode. That is, first of all, the glass electrode must be in contact with the cell and form a high-resistance seal. After the cell seal is completed, the electrode is quickly lifted up so that the cell membrane around the electrode is torn off from the cell due to the upward pulling force. Due to the fluidity of the cell membrane itself, the torn cell membrane will fuse together again to form a vesicle. If a single-layer membrane sheet with the inside facing out is to be obtained, the vesicle needs to be ruptured on the outside of the electrode without affecting the high-resistance sealing state between the electrode and the cell membrane, as well as the membrane sheet on the inside of the electrode.

[0009] There are generally two ways to rupture the vesicles: air exposure and low-concentration calcium ion solution exposure, that is, moving the electrode to change the environmental conditions of the vesicle at the tip of the electrode so that it moves from the extracellular fluid to the air or low-concentration calcium ion solution. The cell membrane will rupture faster in these two environments. The present invention chooses to use the air exposure method because the movement speed of the operating arm is limited in robotic operation. If the low-concentration calcium ion solution exposure method is selected, the vesicle needs to be moved to the calcium ion solution first, and then moved to the intracellular fluid. During the movement, it will also be exposed to the air for a certain period of time, which generally causes the two layers of the vesicle to rupture completely, and it is impossible to obtain a single layer of membrane with the inner side facing outward.

[0010] Therefore, the air exposure method is chosen, that is, the electrode is lifted out of the liquid surface and briefly exposed to the air, so that the vesicles rupture and form a single-layer membrane sheet with the inside facing out. There is no definitive answer to the specific time for the electrode to be lifted out of the liquid surface and exposed to the air. For different cells in different states, the physical properties of their cell membranes are different, and the time required to be exposed in the air is different, which generally takes a few seconds to complete. Since it is difficult to directly see the formed vesicles and their state under a microscope (400×), the resistance value at the tip of the electrode does not change significantly before and after the vesicle ruptures (it always shows a sealed state). Therefore, it is impossible to observe whether the vesicles are ruptured under the microscope or by the resistance value. When the exposure time is too long, the single-layer membrane sheet will also rupture, which is manifested by the decrease in resistance when the electrode is re-entered into the liquid, just like the performance when the electrode is just entered into the liquid. Therefore, it can be proved that the longest time the electrode is exposed to the air. For example, if the single-layer membrane also ruptures when exposed for 5s, you can try to expose it for 4s, so that you can find the longest exposure time, so that the vesicles rupture and the single-layer membrane sheet remains. In this way, the vesicle rupture process can be detected by observing the resistance value at the tip of the electrode.

[0011] S2: Design a fluid replacement device to ensure the rapidity and stability of the fluid replacement process. Since the maximum exposure time of the vesicles to the air is about 3-5 seconds, and the robot arm has a limited movement speed; at the same time, the electrode tip and vesicles are relatively fragile, and it is not suitable to generate large vibrations around them. Therefore, it is necessary to design a fluid replacement device so that the electrode can be removed from the extracellular fluid and then lowered into the intracellular fluid in a relatively short time.

[0012] The present invention designs a rapid liquid replacement device, such as Figure 2 As shown. Since the electrode needs to be moved quickly and stably from the extracellular fluid to the intracellular fluid during the operation, and the electrode can only move up and down within a short time range, the solution directly below the needle tip needs to be replaced quickly and stably. The device is provided with two grooves, both of which are suitable for culture dishes with a diameter of 60 mm. A culture dish containing intracellular fluid is placed in one groove, and a culture dish containing extracellular fluid and cells is placed in the other groove. When preparing for the experiment, the culture dish containing extracellular fluid and cells is placed on the outside, so that the rapid fluid exchange device can be quickly pushed inward after the vesicle is formed. When the device is pushed to the innermost side of the microscope stage, the culture dish containing intracellular fluid is just directly below the electrode. At this time, it is only necessary to quickly lower the electrode.

[0013] S3: Robotic operation to obtain a single-layer membrane with the inside facing out.

[0014] Compared with the prior art, one or more of the above technical solutions have the following beneficial effects:

[0015] 1. The present invention can be applied to nerve cells in brain slices or cells in tissues, and has more practical application value than the robotic inside-out operation of suspended cells. Through the detection of the vesicle rupture process and the design of a rapid liquid replacement device, the robotic inside-out monolayer membrane patch was completed on adherent cells for the first time, which is of great significance for promoting the promotion and application of the inside-out patch clamp technique.

[0016] 2. The reverse verification method for vesicle rupture of the present invention is applicable to a variety of cells, including adherent cells, suspended cells, and nerve cells in brain slices. Different types of cells have different ways of obtaining vesicles, and sometimes it is necessary to add a suction needle to assist in the formation of vesicles, but the reverse verification method is used to obtain the longest time for the exposure of the single-layer membrane sheet to rupture the vesicle. The rapid liquid exchange device designed by the present invention can be customized for culture dishes of different sizes, with low price, fast construction period, and simple equipment requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 It is a schematic diagram of the formation process of the inside-out operation mode in the present invention;

[0019] Figure 2 This is a picture of the use of the rapid liquid changing device of the present invention;

[0020] Figure 3 is a flow chart of step S3 in the present invention;

[0021] Figure 4 is a schematic diagram of HEK293 cells in the present invention under a 40x microscope;

[0022] Figure 5 It is a flow chart of the technical solution of the present invention. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0024] Embodiment 1:

[0025] This embodiment discloses a robotic inside-out patch clamp technique based on vesicle rupture process detection, comprising the following steps:

[0026] S1: By analyzing the mechanism of vesicle formation and rupture, the reverse verification method was used to obtain a method for sealing a monolayer membrane sheet, thereby verifying that a monolayer membrane sheet with the inside facing out was obtained;

[0027] S2: Design a liquid replacement device to ensure the rapidity and stability of the liquid replacement process;

[0028] S3: Robotic operation to obtain a single-layer membrane with the inside facing out.

[0029] The operation flow of step S3 is as follows: Figure 3 As shown, the details are as follows:

[0030] (1) Acquisition of adherent cells.

[0031] The adherent cells used in this embodiment are HEK-293 cells, and the cell line is provided by the Cell Bank of the Chinese Academy of Sciences. After receiving the cell line, it is necessary to digest it, take a small amount of cells for passaging, and freeze the remaining cells. Use trypsin to digest the cells and place them in a 37°C, 5% carbon dioxide incubator for two minutes to digest until the cell adhesion state disappears and becomes round. Use 15% FBS culture medium to repeatedly blow the bottom of the dish to suspend the cells, transfer them into a centrifuge tube and centrifuge. Discard the supernatant, add culture medium to mix, evenly divide the cell suspension into culture dishes, add an appropriate amount of culture medium, and place it in an incubator to complete the passaging. The cells after the passaging can be used for experimental operations after 24 hours. It is better to use cells with a sparser density for experiments, such as Figure 2 shown.

[0032] The method of freezing cells is: after the cell digestion is completed, add the freezing solution to resuspend, put 500μl / tube into the gradient freezing box, and then put it into the -80℃ refrigerator for storage, and put it into liquid nitrogen after 24 hours. The generation number, date, and name need to be marked on the cryopreservation tube. When the cells need to be revived for passaging, take the cryopreservation tube out of the liquid nitrogen, immediately put it into a sealed bag, and thaw it in a water bath. Quickly transfer to a centrifuge tube and centrifuge. Discard the supernatant, add 15% FBS culture medium to resuspend, put it into a 60mm culture dish, add an appropriate amount of 15% FBS culture medium, mix well, and observe under a microscope. Put it in a 37℃ incubator, observe every day, and change the liquid in time (usually 48 hours).

[0033] (2) Electrode preparation.

[0034] The electrode used in this embodiment is drawn from a Sutter borosilicate glass microtube using a Sutter P-97 needle puller. The outer diameter of the glass microtube is 1.5 mm, the inner diameter is 0.86 mm, and the length is 10 cm. In order to reduce noise, the glass microtube needs to be cleaned before drawing the electrode. The specific operation is: soak the glass blank in anhydrous ethanol, treat it with ultrasound for 10 minutes, then wash it with distilled water, and finally place it in a 200°C oven for drying. The diameter of the electrode tip after drawing should be about 1-2 μm.

[0035] (3) Preparation of intracellular and extracellular fluids.

[0036] The intracellular fluid components used in this example are: 75 mM KCl, 10 mM NaCl, 70 mM KF·2H2O, 2 mM MgCl2, 10 mM EGTA, 10 mM HEPES, and the pH is adjusted to 7.2 using KOH; the extracellular fluid components are: 160 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, 10 mM HEPES, and the pH is adjusted to 7.4 using NaOH.

[0037] (4) Sealing cells.

[0038] In this embodiment, the operation process of automated cell sealing is completed through the cooperation of electrophysiological data acquisition and analysis software with non-commercial development software. First, inject an appropriate amount of extracellular fluid into the drawn electrode so that it can submerge the silver wire when installed at the end of the robotic arm, thereby receiving the signal. Take a dish of cultured cells, remove the culture medium, slowly inject 37°C extracellular fluid along the edge, and place it in the groove in the fluid exchange device; take another clean culture dish, inject 37°C intracellular fluid, and place it in another groove in the fluid exchange device. Note that the liquid levels in the two culture dishes need to be as high as possible and close to the edges of the culture dishes. Figure 1 shown.

[0039] Place the liquid exchange device under the microscope, with the culture dish containing cells on the outside and the culture dish without cells on the inside. Control the operating arm to make the electrode drop below the liquid surface, control the air pump to give the electrode a small positive pressure (about 1kPa), and blow air outward to prevent impurities in the liquid from clogging the electrode tip. At this time, the resistance of the glass electrode is about 5MΩ.

[0040] Continue to lower the electrode and use micromanipulation to bring the electrode close to the target cell. When it approaches or touches the target cell, the resistance will increase due to the increase in resistance at the electrode tip. Remove the positive pressure and further apply a smaller positive pressure. The pressure value gradient increases, and a high-resistance seal can be formed between the electrode tip and the membrane, with a resistance of more than 1GΩ. Adjust the air pressure in the electrode to atmospheric pressure to complete the cell sealing operation.

[0041] (5) Vesicle rupture.

[0042] After forming a high-resistance seal, quickly lift the electrode away from the cell to form a vesicle (it may also form a single-layer membrane sheet). After the vesicle is formed, slowly move the electrode up to the highest position below the liquid surface, so that the tip of the electrode remains in the solution, but it can be separated from the solution by slightly moving up and exposed to the air. Quickly lift the electrode into the air, push the fluid exchange device inward, so that the culture dish containing the intracellular fluid is just below the electrode, and then quickly lower the electrode to immerse it in the intracellular fluid. The whole process takes about five seconds. If the electrode resistance still remains in the high-resistance seal state, it means that the inside-out operation mode has been formed.

[0043] (6) Record the signal.

[0044] Give the cell membrane corresponding electrical stimulation and record the voltage and current response signals.

[0045] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a robotic inside-out patch clamp based on the detection of vesicle rupture process, characterized in that: The following steps are involved: S1: By analyzing the mechanism of vesicle formation and rupture, the reverse verification method was used to obtain a method for sealing a monolayer membrane sheet, thereby verifying that a monolayer membrane sheet with the inside facing out was obtained; S2: Design a liquid replacement device to ensure the rapidity and stability of the liquid replacement process; S3: Robotic operation to obtain a single-layer membrane with the inside facing out; In the step S1, the vesicles are ruptured by using an air exposure method, that is, the electrode is lifted out of the liquid surface and briefly exposed to the air, so that the vesicles are ruptured to form a single-layer membrane sheet with the inner side facing outward; When using the air exposure method, the vesicle rupture process is detected by observing the resistance value at the electrode tip, which can be used to measure the maximum time the electrode can be exposed to the air, thereby ensuring that the vesicle ruptures while retaining the unilateral membrane. In step S2, the fluid replacement device includes two grooves, both of which are suitable for culture dishes with a diameter of 60 mm; a culture dish containing intracellular fluid is placed in one of the grooves, and a culture dish containing extracellular fluid and cells is placed in the other groove; After forming a high-resistance seal, quickly lift the electrode away from the cell to form a vesicle; after the vesicle is formed, slowly move the electrode up to the highest position below the liquid surface so that the tip of the electrode remains in the solution, but can be separated from the solution by a slight upward movement and exposed to the air; quickly lift the electrode into the air, and quickly move the fluid exchange device so that the culture dish containing the intracellular fluid is just below the electrode, and then quickly lower the electrode to immerse it in the intracellular fluid. The whole process takes 3-5 seconds; if the electrode resistance still remains in the high-resistance seal state, it means that the inside-out operation mode has been formed.

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